Enhanced activation of natural killer cells using an NK cell activator and a hydrogen peroxide scavenger or inhibitor

ABSTRACT

A method for providing activated natural killer (NK) cells comprising the steps of administering to a population of cells which includes lymphocytes and monocytes, an effective amount of an NK cell activating cytokine or a NK cell activating flavonoid, wherein said NK cell activating cytokine is not IL-2 or IFN-α; and administering a compound effective to inhibit the production or release of hydrogen peroxide selected from the group consisting of histamine, other H 2  receptor agonists, and serotonin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. application Ser. No. 07/843,052,filed Mar. 2, 1992.

This application is a continuation of Application Ser. No. 08/681,108,filed Jul. 22, 1996, now U.S. Pat. No. 6,071,509 which is a continuationof Application Ser. No. 08/287,200, filed Aug. 8, 1994, now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to methods for the enhancedactivation of natural killer (NK) cells, useful for example, in thetreatment of cancer and viral infection. More specifically, the presentinvention relates to the activation of NK cells using a combination of anatural killer cell activator and a hydrogen peroxide inhibitingcompound or scavenger. It also relates to the prevention of inactivationof NK cells.

BACKGROUND OF THE INVENTION

Natural killer (NK) cells are a subset of spontaneously cytotoxiclymphocytes that lytically destroy tumor cells without apparent antigenspecificity or restriction by histocompatibility molecules. Lymphokinesare lymphocyte-derived peptides that modulate immunologic andinflammatory responses by regulating the activity, growth anddifferentiation of a wide variety of leukocyte and nonleukocyte targetcells. Similar factors produced by a variety of cell types, togetherwith lymphokines, are known as cytokines. Several cytokines are known tostimulate proliferation of NK cells and to enhance their cytotoxicactivity.

Interleukin-2 (IL-2), formerly T-cell growth factor (TCGF), is aT-cell-derived cytokine. Since 1985, IL-2 has been used in the treatmentof human neoplasia, mainly in patients with metastasizing solid tumors,such as malignant melanoma and renal cell carcinoma (Rosenberg et al.,N. Engl. J. Med. 316:889-897 (1987); Bukowski et al., J. Clin. Oncol.7:477-485 (1989)), but more recently also in acute myelogenous leukemia(AML) (Foa et al., Br. J. Haematol. 77:491-496.3 (1991)). In the initialstudies, IL-2 was administered together with autologous lymphocytes thathad been treated with IL-2 in vitro, but in recent years IL-2 has morefrequently been administered as a single agent.

The high expectations for the treatment of human cancer using IL-2 werebased on the findings that treatment with IL-2 can induce the regressionof established tumors in several animal tumor models in vivo (Rosenberget al., J. Exp. Med. 161:1169-1188 (1985); Lotre and Rosenberg, inInterleukin-2, K. A. Smith, ed., Academic Press, San Diego, pp. 237-294(1988)). The mechanism underlying this anti-tumor effect of IL-2 hasbeen much debated, but accumulating evidence points to the anti-tumoreffector cell as the natural killer (NK)-cell. Depletion of NK cellsfrom experimental animals eliminates the anti-tumor effect of IL-2 inmany experimental models for tumor growth and metastasis (Mule et al.,J. Immunol. 139:285 (1987)). Further, the only subset of resting humanperipheral blood lymphocytes that carry transducing receptors for IL-2(IL-2R) on the cell surface are NK cells (Caliguri et al., J. Clin.Invest. 91:123-132 (1993)).

IL-2 activates many NK-cell functions, including baseline or “natural”anti-tumor cytotoxicity, antibody-dependent cellular cytotoxicity(ADCC), proliferation, and cytokine production (Trinchieri, Adv.Immunol. 47:187-376 (1989)). Also, IL-2-activated NK cells, frequentlyreferred to as lymphokine-activated killer (LAK) cells, display abroader spectrum of reactivity against human and murine tumor targetcells. Thus, NK cells activated by IL-2 not only kill NK cell-sensitivetumor cells more efficiently, but also kill tumor cells that areinsensitive to the constitutive cytotoxic activity mediated by NK cells.

Recent studies have also shown that IL-2, when combined with histamineor serotonin, augments NK cell cytotoxicity in the presence of monocytesin vitro (Hellstrand et al., J. Immunol. 145(12):4365-4370 (1990) andHellstrand et al., Scand. J. Immunol. 32(2):183-192 (1990)). Thesestudies suggest an interaction between monocytes and NK cells that issubject to regulation by these biogenic amines (Hellstrand et al., J.Interferon Rsch. 12:199-206 (1992). These NK cell regulating mechanismsare thus believed to be of importance to the NK cell mediated responseto metastatic tumors in vivo.

Despite the beneficial effects obtained with IL-2 therapy inexperimental animals and despite the remarkable effects of IL-2 on thekilling activity of human NK cells in vitro, the results of the clinicaltrials of IL-2 in human cancer have, as yet, been disappointing. Only asmall fraction of patients with metastatic melanoma or renal cellcarcinoma show objective regression of tumor burden after treatment withvery high doses of IL-2 (Bukowski et al., J. Clin. Oncol. 7:477-485(1989); whitehead et al., J. Natl. Cancer Inst. 83:1250-1253 (1991)). Inaddition, IL-2 produces severe side effects, including hypotension,fluid retention (“capillary leak syndrome”), fever, lethargy and nausea.

Other interleukins are also known to stimulate NK cell activity. Forexample, IL-12, also known as natural killer cell stimulatory factor(NKSF), is a recently discovered cytokine which has also been reportedto increase NK cell and cytotoxic T lymphocyte activity, T cellproliferation, and the production of interferon-ã. It has been found toenhance the spontaneous cytotoxic activity of peripheral bloodlymphocytes against a variety of tumor-derived target cell lines(Chehimi et al., J. Exp. Med. 175:789-796 (1992)). IL-1 is anothercytokine known to enhance NK cell cytotoxicity.

The interferons consist of a family of secreted proteins with potentantiproliferative and immunomodulatory activities. Theseimmunomodulatory effects include activation of macrophages, augmentationof cellular and humoral immune responses, and enhancement of NK-cellactivity. All three major subtypes of human interferon, i.e.,interferon-á (IFN-á), interferon-â (IFN-â) and interferon-ã (IFN-ã), areknown to enhance NK cell cytotoxicity. Interferon-á (IFN-á) is a majorregulatory factor for NK cells. It has been found to stimulate NK cells(Silva et al., J. Immunol. 125:479-484 (1980)) and augment NK cellcytotoxicity both in vitro and in vivo (Trinchieri, Adv. Immunol.47:187-376 (1989)). Although IFN-á has been shown to be effective withsome neoplasias, the overall results of therapy with high doses of IFN-áhave been disappointing. In addition, patients treated with IFN-á oftenhave acute toxic reactions including fever, chills, myalgias, anorexia,fatigue, headache, nausea and vomiting.

Other known stimulators of NK cell activity include certain flavonoids.The flavonoids are a group of low molecular weight polyphenolicsecondary plant metabolites. Flavone-8-acetic acid has been found topotently augment NK activity in the spleen, liver, lungs, and peritoneum(Wiltrout et al., J. Immunol. 140(9):3261-3265 (1988)).Xanthenone-4-acetic acid (XAA), an analog of FAA, and itsmethyl-substituted derivatives, have also been found to induce NKactivity in vitro (Ching et al., Eur. J. Cancer 27(1):79-83 (1991)).Clinical trials of FAA have been disappointing, however, due tonon-linear pharmokinetics, low dose potency and problems of drugprecipitation.

SUMMARY OF THE INVENTION

The present invention provides a novel method for the activation of NKcells and the prevention of inactivation of these cells by monocytes,using a combination of a lymphokine or other NK cell activator and aperoxide reducing or scavenging compound. The present invention isespecially useful in the treatment of solid tumors and viral infection.

In accordance with one aspect of the present invention, there isprovided a method for providing activated natural killer cellscomprising the steps of administering to a population of cells whichincludes lymphocytes and monocytes, an effective amount of an NK cellactivating compound and a compound effective to inhibit the productionor release of intracellular hydrogen peroxide, provided that when saidNK cell activating compound is IL-2 or IFN-á, said compound effective toinhibit the production or release of intracellular hydrogen peroxide isnot histamine, an H₂ receptor agonist or serotonin.

In a preferred embodiment of the present invention, the compoundeffective to inhibit the production or release of intracellular hydrogenperoxide is histamine, an H₂ receptor agonist or serotonin, and the NKcell activating compound is a cytokine or a flavonoid. In anotherpreferred embodiment, the population of cells is located in vivo. Instill another preferred embodiment, the administration of said NK cellactivating compound and said compound effective to inhibit theproduction or release of intracellular hydrogen peroxide is performedsimultaneously. Alternatively, the administration of said NK cellactivating compound and said compound effective to inhibit theproduction or release of intracellular hydrogen peroxide is performedwithin 24 hours.

In another preferred embodiment of the present invention, the NK cellactivating compound is a cytokine, which is administered in a dose offrom about 1,000 to about 300,000 U/kg/day. In the preferred embodimentwherein the NK cell activating compound is a flavonoid, the flavonoid isadministered in a dose of from about 1 to about 100,000 mg/day. In stillanother preferred embodiment, the compound effective to inhibit theproduction or release of intracellular hydrogen peroxide is administeredin a dose of from about 0.1 to about 10 mg/day.

In accordance with another aspect of the present invention, there isprovided a method for providing activated natural killer cellscomprising the steps of administering to a population of cells whichincludes lymphocytes and monocytes, an effective amount of an NK cellactivating compound and administering a hydrogen peroxide scavenger. Ina preferred embodiment, the hydrogen peroxide scavenger catalyzes thedecomposition of hydrogen peroxide. In a preferred embodiment, thehydrogen peroxide scavenger is catalase, glutathione peroxidase, orascorbate peroxidase. In another preferred embodiment, the NK cellactivating compound is a cytokine or a flavonoid. In still anotherpreferred embodiment, the population of cells is located in vivo.

The administration of said NK cell activating compound and said hydrogenperoxide scavenger is preferably performed simultaneously.Alternatively, the administration of said NK cell activating compoundand said hydrogen peroxide scavenger is performed within 24 hours. In apreferred embodiment, the NK cell activating compound is a cytokine, andthe cytokine is administered in a dose of from about 1,000 to about300,000 U/kg/day. In the preferred embodiment wherein the NK cellactivating compound is a flavonoid, the flavonoid is administered in adose of from about 1 to about 100,000 mg/day. Preferably, the hydrogenperoxide scavenger is administered in a dose of from about 0.1 to about10 mg/day.

DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts the activation of NK cells by catalase andsynergy with IL-2. Culture medium (control; open bars) or IL-2 (10 U/ml;filled bars) was added to enriched human NK cells alone (FIG. 1A) or NKcells admixed with 30% monocytes (FIG. 1B) in the presence of catalaseat the indicated final concentrations. The bars indicate NKcell-mediated killing of target tumor cells (cell lysis % “s.e.m. ofsextuplicate determination).

FIG. 2 graphically depicts the activation of NK cell-mediated clearanceof YAC-1 lymphoma cells in vivo by catalase. Seventy-five thousand YAC-1cells labeled with ⁵¹Cr were injected intravenously into male or female4-6-week-old Swiss Albino mice, together with vehicle (control; openbars) or catalase (100 U/kg; filled bars). Two hours after theinoculation of tumor cells, the mice were sacrificed by cervicaldislocation. The results show retained radioactivity in lung tissue (%of radioactivity retained in lungs at t=0 after injection of labeledtumor cells). Results from 4 separate experiments are shown. Each barrepresents the mean “s.e.m. of 3-5 animals.

FIG. 3 shows the inhibition of IL-2-induced NK cell proliferation andcytotoxicity by monocytes and its reversal by histamine and catalase.Culture medium (open bars), histamine (hatched bars), and catalase(filled bars) were added to enriched NK cells (NK) or a mixture of NKcells and monocytes (NK+MO). FIG. 3A shows NK cell proliferation, andFIG. 3B shows cytotoxicity against K562 target cells.

FIG. 4 illustrates the suppression of NK cell cytotoxicity by hydrogenperoxide and the role of myeloperoxidase. Open symbols represent thecytotoxicity of cells treated with hydrogen peroxide. Filled symbolsrepresent corresponding cells treated with myeloperoxidase.

FIG. 5 illustrates the kinetics of monocyte-induced inhibition of NKcell cytotoxicity. A mixture of NK cells and monocytes were treated withculture medium (open bars), histamine (hatched bars) or catalase (filledbars) at the indicated time points after the start of the microtoxicityassay.

FIG. 6 shows that histamine inhibits the generation of hydrogen peroxidein monocytes. The luminol-enhanced chemiluminescence response ofmonocytes treated with culture medium (solid line) or histamine (dottedline) is shown in FIG. 6A. FIG. 6B shows the response of monocytestreated with sodium azide (control, solid line), or histamine plussodium azide (dotted line).

FIG. 7 shows that histamine H₂-type receptors transduce the effects ofhistamine on the respiratory burst of monocytes. Monocytes were treatedwith histamine plus ranitidine (open circles) or AH20239AA (filledcircles).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for activating NK cells in thepresence of monocytes by using a hydrogen peroxide inhibiting orscavenging compound in combination with a cytokine or other NK cellactivator. The method of the present invention is useful, for example,as a method of inhibiting tumor growth and the formation of metastasesof malignant tumor cells, and in the treatment of viral infection.

In the monovalent pathway of oxygen reduction, superoxide anion (O₂ ⁻)is produced first, followed by the formation of hydrogen peroxide(H₂O₂). Superoxide anion can react with hydrogen peroxide to formhydroxyl radical (OH⁻). These reactive oxygen intermediates (ROI) areproduced by phagocytes such as monocytes and polymorphonuclearneutrophils (PMNs). Hydrogen peroxide produced by monocytes has beenfound to suppress NK cell mediated cytotoxicity. This NK cellcytotoxicity plays an important role in a host's defenses againstarising neoplasms and metastatic tumor cells in vivo. It has now beendiscovered that monocytes suppress NK cell cytotoxicity and that thismonocyte derived suppressive signal effectively down-regulates thecytotoxic and proliferative activities of NK cells. Suppression of NKcells has been found to be halted in the presence of the biogenic amineshistamine and serotonin (Hellstrand et al., J. Immunol. 145:4365-4370(1990)).

It is one of the surprising discoveries of the present invention thatcompounds which reduce the amount of hydrogen peroxide, whenadministered in combination with a cytokine or other compound known tostimulate NK cell activity, act to synergistically stimulate NK cellcytotoxicity in the presence of monocytes; thus, the administration ofscavengers of peroxide, or compounds which inhibit the production orrelease of intracellular peroxide, in combination with a cytokine orother NK cell activator, has been found effective in the treatment ofsolid tumors and viral infection.

Known scavengers of hydrogen peroxide include the enzymes catalase,glutathione peroxidase and ascorbate peroxidase. Compounds which inhibitthe production or the release of intracellular peroxide are alsoeffective in enhancing NK cell activity when administered together withan NK cell activator. Such compounds include serotonin, histamine, andH₂ receptor agonists such as dimaprit.

The present invention therefore provides an effective method forpreventing the inactivation of NK cells and for activation these cells.It also provides a method for treatment of tumors and viral infection,through the administration of compounds which reduce the amount ofhydrogen peroxide, in combination with a cytokine or other compoundknown to stimulate NK cell activity. It is intended that the presentinvention cover the administration of the compounds listed and thosecompounds with similar activity, with the understanding that if thecytokine administered is IL-2 or IFN-á, the inhibitor is not histamine,an H₂ receptor agonist, or serotonin.

Administration of NK Cell Activator and Hydrogen Peroxide Scavenger orInhibitor

The administration of the cytokine or other compound known to enhance NKcell activity, together with the inhibiting or scavenging compoundsdiscussed above, can be by any of a number of methods well known tothose of skill in the art. Such methods include the parenteral deliverythrough intravenous, intraperitoneal, or intramuscular injection. The NKcell activity enhancer and the hydrogen peroxide scavenger can beadministered separately or as a single composition. When administeredseparately, it is contemplated that the NK cell activity enhancer may beadministered either first or last.

The compounds of the present invention may be administered in water withor without a surfactant such as hydroxypropyl cellulose. Dispersions arealso contemplated, such as those utilizing glycerol, liquid polyethyleneglycols, and oils. Antimicrobial compounds may also be added to thepreparations. Injectable preparations may include sterile aqueoussolutions or dispersions and powders which may be diluted or suspendedin a sterile environment prior to use. Carriers such as solvents ordispersion media contain water, ethanol polyols, vegetable oils and thelike may also be added to the compounds of the present invention.Coatings such as lecithins and surfactants may be used to maintain theproper fluidity of the composition. Isotonic agents such as sugars orsodium chloride may be added, as well as products intended to delayabsorption of the active compounds such as aluminum monostearate andgelatin. Sterile injectable solutions are prepared according to methodswell known to those of skill in the art and can be filtered prior tostorage and/or use. Sterile powders may be vacuum or freeze dried from asolution or suspension them. Sustained-release preparations andformulations are also contemplated by the present invention. Anymaterial used in the composition of the present invention should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed.

All preparations may be provided in dosage unit forms for uniform dosageand ease of administration. Each dosage unit form contains apredetermined quantity of active ingredient calculated to produce adesired effect in association with an amount of pharmaceuticallyacceptable carrier.

Although in the Examples which follow the compounds are administered asa single dose, it should be understood that the compounds may beadministered for prolonged periods of time. Typically, the treatment maybe administered for periods up to about one week, and even for periodslonger than one month. In some instances, the treatment may bediscontinued and then resumed at a later time. A daily dose may beadministered as a single dose, or it can be divided into several doses,especially if negative effects are observed. In addition, the compoundsof the present invention can be administered as a single composition, orseparately. If administered separately, the compounds should be given onthe same day, such that the activation of NK cells by the lymphokine orother compound is enhanced.

Preferred dosage range can be determined using techniques known to thosehaving ordinary skill in the art. IL-1, IL-2 or IL-12 can beadministered in an amount of from about 1,000 to about 300,000 U/kg/day;more preferable, the amount is from about 3,000 to about 100,000U/kg/day, and even more preferably, the amount is from about 5,000 toabout 20,000 U/kg/day.

IFN-á, IFN-â, and IFN-ã can also be administered in an amount of fromabout 1,000 to about 300,000 U/kg/day; more preferable, the amount isfrom about 3,000 to about 100,000 U/kg/day, and even more preferably,the amount is from about 10,000 to about 50,000 U/kg/day.

Flavonoid compounds can be administered in an amount of from about 1 toabout 100,000 mg/day; more preferable, the amount is from about 5 toabout 10,000 mg/day, and even more preferably, the amount is from about50 to about 1,000 mg/day.

Compounds which inhibit the release or formation of intracellularhydrogen peroxide, or scavengers of hydrogen peroxide, can beadministered in an amount of from about 0.1 to about 10 mg/day; morepreferable, the amount is from about 0.5 to about 8 mg/day, and evenmore preferably, the amount is from about 1 to about 5 mg/day. However,in each case, the dose depends on the activity of the administeredcompound. The foregoing doses are appropriate for histamine, catalaseand for H₂ receptor agonists. Appropriate doses for any particular hostcan be readily determined by empirical techniques well known to those ofordinary skill in the art.

The method of the present invention may be utilized alone or incombination with other anti-cancer therapies, as determined by thepractitioner.

Monocyte-Induced Inhibition of NK Cells: Reversal by Catalase and theRole of Reactive Oxygen and Nitrogen Species

To investigate the effects of hydrogen peroxide scavengers on theactivation of NK cells by cytokines, we studied the effects of catalase,a heme containing enzyme that catabolizes hydrogen peroxide (H₂O₂) tooxygen and water, on human NK cell-mediated killing of tumor cells invitro and on NK cell function in mice in vivo. These experiments aredescribed below in Examples 1 and 2. The following examples are merelyillustrative of the present invention, and are not intended to limit theinvention in any way.

EXAMPLE 1

Using blood obtained from a healthy human blood donor, we studied theeffects of catalase on NK cell-mediated killing of tumor cells. K562cells, from an NK sensitive erythroleukemic cell line, were used astarget cells in all experiments. Washed cells (10×10⁶ cells/ml) wereincubated with ⁵¹Cr (Amersham) at a concentration of 150 FCi/ml cellsuspension for 2-4 hours. After centrifugation and resuspension in cellculture medium, 10⁴ cells in 50 FL portions were added to the effectorcells in microplate wells.

In the first set of experiments, human NK cells alone (1.5×10⁵cells/well) were added to the K562 target cells. The combined cells werethen exposed to culture medium (control) or human recombinant IL-2(EuroCetus, Amsterdam, The Netherlands) at a final concentration of 10U/ml. The cells were then exposed to catalase (Boehringer-Mannheim) atconcentrations of 0 to 100 U/ml. These same conditions were repeatedusing human NK cells admixed with 30% human peripheral blood monocytes,recovered by centrifugal elutriation, added to the target K562 cells.

After incubation at 37EC for 16 hours, supernatant fluids were collectedby a tissue collecting system (Amersham) and assayed for radioactivityin a gamma counter. Maximum ⁵¹Cr release was determined in target cellcultures treated with Triton-X. NK cell cytotoxicity was calculated ascell lysis % according to the following formula:${{cell}\quad {lysis}\quad \%} = {100 \times \frac{{{experimental}\quad {release}} - {{spontaneous}\quad {release}}}{{{maximum}\quad {release}} - {{spontaneous}\quad {release}}}}$

The results of these studies are illustrated in FIG. 1. FIG. 1A showsthat catalase does not affect the level of NK-cell-mediated killing oftumor cells in the absence of monocytes, regardless of whether the NKcells are unactivated or activated by IL-2. FIG. 1B shows that theanti-tumor activity of unactivated NK cells is suppressed by thepresence of monocytes. Further, catalase, at final concentrationsexceeding 10 U/ml, reverses this suppressive signal. FIG. 1A furthershows that IL-2 does not significantly activate NK-cell cytotoxicityagainst tumor cells in the presence of monocytes unless catalase ispresent.

Effects of Catalase and Other Scavengers

To study the relationship between the monocyte-derived inhibitory signaland the respiratory burst activity of monocytes, we added catalase andvarious other scavengers of reactive oxygen metabolites to NK cells,alone or admixed with monocytes. We then measured the cytotoxicity ofthe NK cells against NK cell sensitive K562 target cells as describedabove. The results of this testing are shown in Table I.

TABLE I Effect of scavengers of reactive oxygen and nitrogen metaboliteson monocyte-induced inhibition of NK-cell cytotoxicity. exp. cell lysis%^(a) no. treatment conc. MO medium histamine 1 catalase 0 − 55″3 53″2 ″20 U/ml − 60″3 52″4 ″ 0 + 19″2 57″1 ″ 2.5 U/ml + 21″1 55″2 ″ 5 U/ml +34″2 55″2 ″ 10 U/ml + 49″1 52″2 ″ 20 U/ml + 57″2 50″4 SOD 200 U/ml −52″2 54″2 ″ 200 U/ml + 1.3″2  54″2 2 taurin 0 − 72″2 70″3 ″ 10⁻³ M −70″4 75″4 ″ 0 +  6″2 63″4 ″ 10⁻³ M +  6″1 64″3 3 deferox 0 − 79″4 78″2 ″10⁻⁴ M − 81″2 74″5 ″ 0 +  9″3 62″2 ″ 10⁻⁴ M + 12″3 60″2 4 mannitol 0 −77″4 69″2 ″ 3 ×10⁻⁴ M − 73″3 68″3 ″ 0 + 19″2 64″4 ″ 3 × 10⁻⁴ M + 20″167″2 5 L-NMMA 0 − 58″1 51″2 ″ 2 × 10⁻4 M − 61″3 54″2 ″ 0 + 12″1 45″2 ″ 2× 10⁻⁴ M + 11″1 45″3 ^(a)NK-cell-enriched lymphocytes treated withculture medium (control), histamine (10⁻⁴ M), and catalase, SOD, taurin,mannitol, deferoxamine (deferox), or L-NMMA at indicated finalconcentrations in the presence (MO+) or absence (MO−) of monocytes. Allcomponents were added at the onset of a microcytotoxicity assay againstK 562 target cells. Data are cell lysis % ″ s.e.m. of sextuplicates andshow results from five separate experiments.

Catalase, which effectively degrades H₂O₂, had no effect on thecytotoxicity of NK cells in the absence of monocytes but was found tocompletely abrogate the monocyte-induced inhibition of baseline NK cellcytotoxicity. Catalase was effective at concentrations exceeding 5 U/ml.Histamine (histamine dihydrochloride; Sigma) at concentrations exceeding10⁻⁷ M abrogated the monocyte induced suppression of NK cells but wasineffective in the presence of catalase.

It was also discovered that superoxide dismutase (SOD), a scavenger ofO₂ ⁻, did not alter the suppressive effect of monocytes on NK cells overa wide range of concentrations. Similarly, taurin, a scavenger of HOCl⁻,and scavengers of OH⁻ such as mannitol and deferoxamine, wereineffective at reducing the suppressive effects of monocytes on NKcells.

Further, monocytes and macrophages produce reactive nitrogenintermediates of which nitric oxide (NO) is the ultimate effectormolecule. To study whether NO induction in monocytes contributed to NKcell inhibition, we used a NO synthetase inhibitor,N-monomethyl-L-arginine (L-NMMA). This compound, used at concentrationssufficient to inhibit induction of NO in monocytes, did not alter thesuppression of NK cell function by monocytes. These results are alsoshown in Table I.

Thus, we have concluded that NK cell-mediated cytotoxicity is suppressedby H₂O₂ produced by monocytes. This suppression of NK cell-mediatedcytotoxicity induced by H₂O₂ is abrogated by the presence of catalase orhistamine. In addition, it was discovered that human NK cells do notrespond to IL-2 unless the monocyte-derived H₂O₂ is scavenged bycatalase or by some other scavenger.

In vivo Effects of Hydrogen Peroxide Scavengers

To study whether the regulatory effects on human NK cell functioninduced by catalase in vitro are of importance for NK cell-mediatedkilling of tumor cells in vivo, experiments were performed in whichcatalase was injected intravenously to mice shortly before intravenousinoculation of NK cell-sensitive tumor cells. These experiments aredescribed below in Example 2.

EXAMPLE 2

Seventy-five thousand ⁵¹Cr-labeled, NK cell-sensitive YAC-1 mouselymphoma cells were injected intravenously into male or female 4 to 6week old Swiss Albino mice, together with vehicle (control) or 100 U/kgcatalase. Two hours after inoculation with the tumor cells, the micewere sacrificed by cervical dislocation. The lungs were removed andplaced in test tubes in a gamma-counter, and the radioactivity in thelung tissue was measured. The radioactivity in lung tissue is an inversemeasure of NK cell-mediated killing of tumor cells in vivo (see Hanna etal., JNCI 65:801 (1980)), and is expressed as a percent of the ⁵¹Cr thatis retained in lungs immediately after inoculation of radiolabeled tumorcells.

The results of this testing are shown in FIG. 2. The data in FIG. 2represent four separate experiments. Each bar represents the retainedradioactivity in lung pairs (mean “s.e.m. of 3-5 animals). Consistently,it was found that treatment with catalase augmented the NK cell-mediatedkilling of YAC-1 lymphoma cells in vivo.

To determine whether the NK cell-mediated killing of tumor cells isenhanced using other NK cell activators and other peroxide scavengers,the experiments described below are performed.

EXAMPLE 3

The experiment described in Example 1 is repeated using IL-12 as the NKcell activator. Similar results are obtained.

EXAMPLE 4

The experiment described in Example 1 is repeated using IFN-á as the NKcell activator. Similar results are obtained.

EXAMPLE 5

The experiment described in Example 1 is repeated using FAA as the NKcell activator. Similar results are obtained.

EXAMPLE 6

The experiment described in Example 2 is repeated using glutathioneperoxidase as the hydrogen peroxide scavenger. Similar results areobtained.

EXAMPLE 7

The experiment described in Example 2 is repeated using ascorbateperoxidase as the hydrogen peroxide scavenger. Similar results areobtained.

Inhibition of IL-2 Induced NK Cell Functions by Monocytes: Reversal byCatalase and Histamine

IL-2 activates NK cell mediated cytotoxicity and induces proliferationof the resting population of NK cells. Elutriated monocytes effectivelyinhibit the IL-2 induced proliferation of enriched NK cells as well asthe activation of NK cell cytotoxicity. To show that histamine, acompound we have discovered to suppress the generation of H₂O₂ inmonocytes, and catalase, a scavenger of H₂O₂, reverse themonocyte-induced inhibition, the following experiments were performed.

EXAMPLE 8

Cell culture medium (control), histamine (10⁻⁴ M), or catalase (20U/ml), was added to either enriched NK cells alone or a mixture of NKcells and monocytes in microplates (1.5×10⁵ cells/well). Each group ofcells then received 50 U/ml human recombinant IL-2 and were allowed toincubate for 48 hours. During the last 8 hours of incubation, cells werepulsed with ³H-methyl-thymidine (specific activity 2 Ci/mole; NewEngland Nuclear Corp.; 1 FCi/2×10⁵ cells). Following incubation, thecells were collected on glass fiber filters with an automatic cellharvester and cell-incorporated ³H-methyl-thymidine was estimated byliquid scintillography.

The results are shown in FIG. 3A, which illustrates NK cellproliferation, as reflected by ³H-methyl-thymidine incorporation aftertreatment with IL-2. The bars represent cpm×10³ (proliferation)±s.e.m.of sextuplicates. The results show that monocytes inhibit theproliferation of NK cells induced by IL-2. Both histamine and catalaseeffectively reverse this monocyte-induced inhibition.

To show the inhibitory effect of monocytes on IL-2 induced NK cellcytotoxicity, and its reversal by histamine and catalase, the followingexperiment was performed.

EXAMPLE 9

Cell culture medium (control), histamine (10⁻⁴ M), or catalase (20U/ml), was added to either enriched NK cells alone or a mixture of NKcells and monocytes (1.5×10⁵ cells/well). These mixtures were thenincubated with K562 target cells in sextuplicate in microplates in atotal volume of 200 Fl and assayed for microcytotoxicity. Afterincubation at 37EC for 16 hours in the presence of culture medium(control) or IL-2 (50 U/ml), supernatant fluids were collected andassayed for radioactivity as described above in connection withExample 1. The results are illustrated in FIG. 3B.

FIG. 3B shows the cytotoxicity of the respective cell mixtures againstK562 target cells. The bars represent percent cell lysis ±s.e.m. ofsextuplicates. Again, it is clear from this data that monocytes inhibitthe cytotoxicity of NK cells induced by IL-2. Both histamine andcatalase effectively reverse this monocyte-induced inhibition.

Reconstitution of Monocyte Induced Inhibition by H₂O₂

The finding that catalase, but not scavengers of O₂ ⁻ or of OH⁻,reversed the suppression of NK cells by monocytes suggests that H₂O₂, ormetabolites of this compound, is essential for expression of theinhibitory signal. We therefore studied whether hydrogen peroxide couldreconstitute the inhibitory effects of monocytes on NK cells.

EXAMPLE 10

Culture medium (control) or H₂O₂ at concentrations between 0-10 FM wasadded to NK cell enriched lymphocytes for assay of cytotoxicity against⁵¹Cr K562 target cells as described above. Addition of H₂O₂ to enrichedNK cells effectively suppressed NK cell cytotoxicity. The results ofthis testing are shown in FIG. 4, which shows the cell lysis % ±s.e.m.of sextuplicates. The ED₅₀ of H₂O₂ was approximately 2×10⁻⁶ M, as seenin FIG. 4.

It was also discovered that catalase (20 U/ml), but not histamine,completely reversed the inhibition of NK cells induced by H₂O₂ (data notshown).

Role of MPO

To study whether H₂O₂ alone or its reactive metabolites mediated theinhibitory effect of exogenous H₂O₂ on NK cells, myeloperoxidase (MPO),a monocyte-derived enzyme that forms toxic hypohalous acids such as HOClfrom H₂O₂, and halides and OH⁻ from H₂O₂ and ferrous iron, was added toenriched NK cells, alone or together with H₂O₂. If radicals such ashypohalous acids contributed to the NK cell inhibitory signal, it wasexpected that MPO would potentiate the suppressive effect of H₂O₂ onenriched NK cells. This testing is described below in Example 11.

EXAMPLE 11

MPO (100 U/ml) and H₂O₂ at concentrations between 0-10 FM were added toNK cell enriched lymphocytes for assay of cytotoxicity against ⁵¹Cr K562target cells as described above. The results of this testing are shownin FIG. 4, which shows the cell lysis % ±s.e.m. of sextuplicates.

MPO did not potentiate the suppressive effect of H₂O₂ on NK cells. Itwas found that addition of MPO slightly but significantly scavenged H₂O₂in these experiments.

These results, along with the finding the mannitol, taurin anddeferoxamine, all of which are scavengers of MPO catalyzed products, didnot affect the inhibition of NK cells by monocytes suggested that theinhibitory signal is independent of MPO activity.

Kinetics of the Monocyte Derived NK cell Inhibitory Signal

To assess when the inhibitory signal is conveyed from monocytes to NKcells, experiments were performed in which catalase or histamine wereadded to mixtures of monocytes and NK cells at various time points afterthe beginning of the microcytotoxicity assay against K562 target cells.

EXAMPLE 12

A mixture of enriched NK cells and monocytes were treated with culturemedium (control), histamine (10⁻⁴ M), or catalase (20 U/ml) and assayedfor microcytotoxicity against target K562 cells as described above inconnection with Example 1. It was found that catalase and histamine wereeffective in inhibiting the NK cell suppressive signal only when thesecompounds were added within the first hour of incubation of monocyteswith NK cells. The results of this testing are shown in FIG. 5. The datashown is cell lysis % ±s.e.m. of sextuplicates.

In additional experiments, NK cell enriched lymphocytes were pretreatedin petri dishes with culture medium (control), catalase or histamine, inthe concentrations indicated below in Table II, in the presence orabsence of monocytes. After 1 hour incubation, nonadherent lymphocyteswere recovered, washed twice, and assayed for cytotoxicity as describedabove. We wished to determine whether the NK cell inhibitory signal wasreversible by removal of monocytes and removal of monocyte-derivedproducts. It was found that lymphocytes recovered from monocyte/NK cellmixtures pretreated with catalase or histamine were more cytotoxicagainst K562 target cells than control mixtures pretreated with mediumonly. The results of this testing are shown below in Table II. Data arecell lysis % ±s.e.m. of sextuplicates and are the results of 2 separateexperiments.

TABLE II Irreversible inhibition of NK-cells by monocytes and H₂O₂. exp.no. pretreatment conc. MO cell lysis %^(a) 1 medium − 66″3 histamine10⁻⁵ M − 59″3 catalase 20 U/ml − 67″2 medium + 14″1 histamine 10⁻⁵ M +51″2 catalase 20 U/ml + 48″3 2 medium − 59″3 H₂O₂ 1.5 × 10⁻⁶ M − 26″2 ″3 × 10⁻⁶ M − 12″1 ″ 6 × 10⁻⁶ M −  2″1 ^(a)NK-cell-enriched lymphocyteswere pretreated in petri dishes with culture medium (control),histamine, or catalase at indicated final concentrations in the presence(MO+) or absence (MO−) of monocytes. Thereafter, nonadherent lymphocyteswere recovered, washed twice and assayed for cytoxicity against K562target cells. Data are cell lysis % ″ s.e.m. of sextuplicates and showresults from two separate experiments.

These data show that the inhibition of NK cells is evoked within thefirst hour of incubation with monocytes and that the inhibition is notreversible by removal of monocytes or monocyte derived factors. Toconfirm this finding, enriched NK cells were treated with H₂O₂ for 20minutes followed by extensive washing and assay for cytotoxicity.Pretreatment with H₂O₂ at micromolar concentrations was sufficient toeffectively inhibit NK cell cytotoxicity. The results of this testingare shown in Table II.

Histaminergic Regulation of the Respiratory Burst of Monocytes

Histamine has been reported to affect several functions ascribed tomonocytes and macrophages, but effects of histamine on the respiratoryburst of monocytes have remained unknown. To determine these effects, wefirst tested whether histamine could act as a scavenger of H₂O₂ or itsradical metabolites in a cell free system. We then studied the effectsof histamine and H₂R-interactive compounds on the respiratory burstactivity of monocytes. These experiments are described in the followingExamples.

EXAMPLE 13

Chemiluminescence (CL) of cells was recorded at 37EC in a 6-channelBiolumat LB 9505 (Berthold Co., Wildbad, Germany) using 4 mlpolypropylene tubes as described by Lock et al. Anal. Biochem. 173:450(1988). The reaction mixture contained 0.8 ml elutriated monocytes(5×10⁶ cells/ml). The tubes were allowed to equilibrate for 5 minutes at37EC before formylmethionyl-leucyl-phenylalanine (fMLP; Sigma; 10⁻⁷ Mfinal concentration) and luminol (Sigma; 10⁻⁶ M) were added and lightemission recorded. Formylmethionyl-leucyl-phenylalanine was dissolved to10⁻² M in dimethyl sulfoxide and subsequently diluted in Krebs-Ringerphosphate buffer supplemented with glucose (10 mM), Ca²⁺ (1 mM), andMg²⁺ (1.5 mM). Luminol was dissolved in 0.1 mM NaOH to 5×10⁻² M andfurther diluted in Krebs-Ringer phosphate buffer.

The CL recorded with H₂O₂ and/or MPO (10 Fg/ml) or H₂O₂ and horseradishperoxidase (HRP; Calbiochem, La Jolla, Calif.) was unchanged byhistamine (10⁻⁴ M). Further, we used an assay system in which lysis ofelutriated , ⁵¹Cr -labelled RBC was measured in microplates. Addition ofH₂O₂ (5×10⁻⁵ M) to 10⁵ RBC induced lysis of approximately 50% of RBC.Histamine (10⁻⁴ M) did not alter the level of RBC killing induced byH₂O₂. It is therefore concluded that histamine is not a scavenger ofH₂O₂ or its radical metabolites.

EXAMPLE 14

In a second set of experiments, effects of histamine and H₂R-interactivecompounds on the respiratory burst activity, as measured by theluminol-enhanced CL response of enriched, elutriated monocytes, werestudied. Monocytes were treated with culture medium (control), histamine(10⁻⁵ M), sodium azide (10⁻⁵ M), or histamine and sodium azide asdescribed below. Emission of CL was recorded after addition of fMLP attime=0.

It was found that histamine effectively inhibited both the burstactivity of unstimulated monocytes and the induction of burst by fMLP.The results of this are shown in FIG. 6A. The inhibitory effect ofhistamine was dose dependent at final histamine concentrations of10⁻⁴-10⁻⁷ M.

To assess whether histamine acted by inhibiting the generation of H₂O₂or by reducing the availability of peroxidase, we next studied theeffects of histamine in monocytes treated with sodium azide to inhibitendogenous myeloperoxidase (MPO) and with exogenous, azide-insensitiveperoxidase (HRP) in excess. Histamine inhibited the fMLP induced CLresponse also in this type of assay, showing that histamine specificallyinhibits the formation of H₂O₂ in monocytes. The results of this testingare shown in FIG. 6B.

Dimaprit, (SK&F, Hertfordshire, England), a specific H₂R agonist,mimicked the effect of histamine on the respiratory burst of monocytes.In contrast, nor-dimaprit, (SK&F, Hertfordshire, England), an H₂Rinactive structural analog of dimaprit, was ineffective. A strikingdifference between histamine and dimaprit was that whereas histamineblocked respiratory burst activity within seconds, the effect ofdimaprit was not maximal until after 10-15 minutes of incubation (datanot shown).

FIG. 7 shows that the effects of histamine were entirely blocked bysimultaneous treatment with the specific H₂R antagonist ranitidine(Glaxo). To exclude non-specific effects of ranitidine, we used aranitidine analog (AH20239AA; C₁₃H₂₂O₄; Glaxo) in which the thioether ofranitidine is replaced by an ether, thereby strongly reducing its H₂Rantagonist properties. In these experiments, monocytes were treated withhistamine (10⁻⁵ M) together with ranitidine or AH20239AA at finalconcentrations indicated in FIG. 7. All cells were treated with fMLP attime=0. Peak CL recorded in untreated monocytes (control) was 2.5×10⁷cpm.

The chemical control to ranitidine, AH20239AA, was more than 100-foldless potent than ranitidine in antagonizing the effects of histamine onthe suppression of NK cell function by monocytes, as well as theinhibition of respiratory burst activity by histamine, as shown in FIG.7. The effects of histamine on the respiratory burst activity ofmonocytes, therefore, are specifically transduced by H₂R.

Conclusion

We have discovered that hydrogen peroxide is a pivotal mediator ofmonocyte-derived, NK cell suppressive signal. The inhibitory effectiveof hydrogen peroxide on NK cells was not catalyzed by the addition ofMPO, thus demonstrating that the MPO activity is not required to mediateNK cell inhibitory signals. Further, scavengers of MPO catalyzedradicals do not affect the inhibition of NK cell function induced bymonocytes.

It is clear from our results that histamine, serotonin, or other H₂receptor agonists, acting via monocyte H₂ receptors, inhibit thegeneration of reactive oxygen products by monocytes, and thereby inhibitthe NK cell suppressive signal. It is clear that scavengers of hydrogenperoxide also act to inhibit the NK cell suppressive signal.

We have thus shown that treatment with a combination of an NK cellactivating cytokine or other compound and a hydrogen peroxide scavengeror inhibiting compound in the presence of monocytes prevents theinactivation of NK cells and enhances NK cell cytotoxicity against tumorcells. These are unexpectedly superior results, since under similarcircumstances, NK cell activators alone had no such beneficial effect.Of particular importance is that the potentiation of the anti-tumoreffect of the NK cell activators induced by the concomitant treatmentwith a peroxide scavenger or inhibiting compound permits a reduction inthe high doses of lymphokines which are used in cancer therapy.Advantageously, high dose treatments of lymphokines and the accompanyingserious side effects can be eliminated by the method of the presentinvention.

What is claimed is:
 1. A method for providing activated natural killer(NK) cells comprising the steps of: administering to a population ofcells which includes lymphocytes and monocytes, an effective amount ofan NK cell activating cytokine or a NK cell activating flavonoid,wherein said NK cell activating cytokine is not IL-2 or IFN-α; andadministering a compound effective to inhibit the production or releaseof hydrogen peroxide selected from the group consisting of histamine,other H₂ receptor agonists, and serotonin.
 2. The method of claim 1,wherein said population of cells is located in vivo.
 3. The method ofclaim 1, wherein the administration of said NK cell activating cytokineor said flavonoid and said compound effective to inhibit the productionor release of intracellular hydrogen peroxide is performedsimultaneously.
 4. The method of claim 1, wherein the administration ofsaid compound effective to inhibit the production or release ofintracellular hydrogen peroxide is performed within 24 hours of theadministration of said NK cell activating cytokine or flavonoid.
 5. Themethod of claim 2, wherein said cytokine is administered in a dose offrom about 1,000 to about 300,000 U/kg/day.
 6. The method of claim 2,wherein said flavonoid is administered in a dose of from about 1 toabout 100,000 mg/day.
 7. The method of claim 2, wherein said histamine,other H₂-receptor agonist or serotonin is administered in a dose of fromabout 0.1 to about 10 mg/day.
 8. A method for providing activatednatural killer (NK) cells comprising the steps of: administering to apopulation of cells which includes lymphocytes and monocytes, aneffective amount of an NK cell activating cytokine or a NK cellactivating flavonoid; and administering a hydrogen peroxide scavenger.9. The method of claim 8, wherein said hydrogen peroxide scavengercatalyzes the decomposition of hydrogen peroxide.
 10. The method ofclaim 9, wherein the hydrogen peroxide scavenger is selected from thegroup consisting of catalase, glutathione peroxidase, and ascorbateperoxidase.
 11. The method of claim 8, wherein said population of cellsis located in vivo.
 12. The method of claim 8, wherein theadministration of said NK cell activating cytokine or said flavonoid andsaid hydrogen peroxide scavenger is performed simultaneously.
 13. Themethod of claim 8, wherein the administration of said NK cell activatingcytokine or said flavonoid and said hydrogen peroxide scavenger isperformed within 24 hours.
 14. The method of claim 11, wherein saidcytokine is administered in a dose of from about 1,000 to about 300,000U/kg/day.
 15. The method of claim 11, wherein said flavonoid isadministered in a dose of from about 1 to about 100,000 mg/day.
 16. Themethod of claim 11, wherein said hydrogen peroxide scavenger isadministered in a dose of from about 0.1 to about 10 mg/day.